Apoptosis, or programmed cell death, is a fundamental biological process to protect cells from DNA damage. Defects in the genes involved in regulating apoptosis are frequently found in tumors. In mammalian cells, apoptosis induced by DNA damage is regulated through mitochondria by the Bcl-2 family of proteins. However, the mechanism by which DNA damage initiates apoptosis remains to be fully characterized. Our recent studies identified a novel Bcl-2 family protein called PUMA, which appears to play an important role in cell death. Induction of PUMA triggered a rapid and profound apoptotic response in human cancer cells. PUMA functions through other Bcl-2 family proteins including Bax, Bcl-2 and Bcl-XL. PUMA is a direct target of p53, which plays a central role in DNA damage response. PUMA can also be induced by chemotherapeutic drugs, such as the DNA damaging agent adriamycin, in a p53-dependent manner. Furthermore, deletion of PUMA in colon cancer cells by homologous recombination led to a markedly decreased apoptotic response to p53 and adriamycin. Based on these observations, we propose to test the hypothesis that PUMA mediates initiation of DNA damage-induced apoptosis in human cancer cells: Aim l. To define the mechanism of PUMA-mediated apoptosis initiation in human cancer cells; Aim 2. To determine if PUMA is activated through the p53-regulated DNA damage response pathway; Aim 3. To investigate if the apoptotic response to DNA damage is abrogated in PUMA-deficient cells.